In Vivo Gene Transfer to Mouse Spermatogenic Cells: MATERIALS AND METHODS(2)

Four plasmids containing the firefly luciferase (luc) gene were investigated for the luciferase reporting assay. Basic-luc contains only the luciferase gene without any promoter element (PGV-B; Toyo Ink Mfg. Co. Ltd., Tokyo, Japan). SV40 E/P-luc and SV40 P-luc contain the luciferase gene linked to both the enhancer and promoter regions of the SV40 early gene, and the SV40-promoter region alone, respectively (PGV-C, PGV-P; Toyo Ink Mfg. Co. Ltd.). Prm E/SV40 P-luc contains the luciferase gene driven by the SV40 early promoter region and the TATA-less prm-1 enhancer upstream sequence ranging from -560 to -33, which was isolated by PCR using two primers, 5′-GTCTAGTAATGTCCAACACC and 5′-GATACTAGTGGCCCCTAGGA. The Renilla luciferase gene under control of the CMV promoter (pRL-CMV; Promega, Madison, WI) was cotransfected in each assay to normalize the differences in transfection efficiency between the assays. antibiotic levaquin

All the plasmid DNAs for injection were purified with Qiagen Maxi columns (Qiagen Inc., Chatworth, CA) and dissolved in HBS buffer (20 mM Hepes, 150 mM NaCl; pH 7.4) at concentrations of 1 ^g/^l for CMV-lacZ and Prm-lacZ, and 0.5 ^g/^l for the four luciferase plasmids, in which pRL-CMV was added at the concentration of 0.1 ^g/^l and cotransfected.


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